Coxiella burnetii is an obligate intracellular bacterial pathogen that is typically acquired through aerosol exposure. It is the etiologic agent of acute Q fever and chronic diseases such as endocarditis, hepatitis, and chronic fatigue. The acute form is typically a self-limiting flu-like illness that ranges from sub-clinical to severe. Q fever endocarditis has been implicated as a major cause of non-culturable endocarditis. C. burnetii is environmentally stable, has a low infectious dose, and has previously been developed as a weapon. These characteristics have led to it being listed as a Select Agent by the CDC as a potential bioterror threat. Normally however, animal populations (particularly ruminants) are the reservoirs for C. burnetii shedding environmentally stable infectious particles that can infect humans. As such, this zoonotic pathogen poses a significant risk to persons involved in the cattle, sheep, and goat industries. Initial infection occurs in lung alveolar macrophages. After infecting the host cell, C. burnetii replicates in vacuoles that retain many of the features of mature phagolysosomes. The molecular mechanisms used by C. burnetii to parasitize their host cells are largely unknown. The genome of C. burnetii (Nine Mile Phase I strain) has been sequenced and contains genes homologous to the type IV secretion system (TFSS) of Legionella pneumophila, suggesting that C. burnetii possesses a specialized secretory pathway for interacting with host cells. However, little is known about the TFSS of C. burnetii or its possible role during the infectious cycle. Our central hypothesis is that the C. burnetii TFSS is necessary for bacterial survival, replication and/or dissemination. The overall goals of this project it to determine whether C. burnetii produces proteins that can act as a TFSS and to examine the temporal expression of these proteins. The goals will be achieved by accomplishing the following specific aims: Specific aim 1 - Characterize the expression of TFSS mRNA synthesized by C. burnetii during infection of host cells. We will use reverse-transcriptase PCR (RT-PCR), real time RT-PCR, and primer extension as tools to define the expression of the C. burnetii TFSS homologs in early, mid, or late stages of infection in vitro. Specific aim 2 - Characterize the protein expression and sub-cellular localization of the C. burnetii TFSS homologs during infection. We will accomplish this by producing specific antisera to recombinant TFSS homologs and using these probes to assess protein expression in the bacteria during infection. Characterization of the C. burnetii TFSS during infection is a critical first step towards understanding the mechanisms used by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease. Coxiella burnetii is an obligate intracellular bacterium and the causative agent of acute Q fever and chronic diseases. It is a zoonotic pathogen, which has been designated a Category B level Select Agent by the CDC. Very little is known about the molecular interactions of C. burnetii and its host cell. In the current proposal we will characterize the C. burnetii type IV secretion system (TFSS). Considering the established role of the TFSS in infection and development of other bacteria, these studies will likely lead to the identification of unique diagnostic, therapeutic, and vaccine targets for Q-fever detection and intervention.